CA1192821A

CA1192821A - High glucose-determining analytical element

Info

Publication number: CA1192821A
Application number: CA000425034A
Authority: CA
Inventors: Shuenn-Tzong Chen; Mark Sherwood; Mary E. Warchal
Original assignee: Miles Laboratories Inc
Current assignee: Bayer Corp
Priority date: 1982-06-14
Filing date: 1983-03-31
Publication date: 1985-09-03
Also published as: IE55165B1; ES8405153A1; JPS596900A; EP0101799B1; ZA832503B; FI832096L; IL68312A0; ES523242A0; IN159163B; MX156030A; EP0101799A1; DK158645B; DK270683D0; FI77894B; US4427770A; FI77894C; IE831383L; NO831986L; JPH0355120B2; AU540228B2

Abstract

ABSTRACT

An analytical element for quantitatively determining high levels of glucose in blood is made by (a) impregnating a carrier with a first-solution, having dissolved therein 4-aminoantipyrine and 3-hydroxy-2,4,6-triiodobenzoic acid or 3,5 dichloro-2-hydroxy-benzene sulfonic acid or salts thereof, a glucose oxidase and a peroxidase, and drying the carrier; and (b) applying to the carrier a second solution of a film-forming agent in a volatile solvent, and drying to remove the volatile solvent and leave a film over the dried first impregnant.

Description

-- 2 ~
BACKGROUMD OF T~IE II~VENTION

1. FIELD OF THE INVENTION

The present invention relates g~nerally to the field of diagnostic tests and, more particularly, to te6t devices and elements useful in the.qualltit:ative determination of ~n analyte havint3 a high glucosq content.

2. BRIEF DESCRIPTION OE' T~IE PRIOR ART

~ est devices in th~ form of test strips a~d similar solid phase analytical e~ements have become commonplace in the analysis of various types of samples, particularly biological fluids. Test strips designed for detecting sugar, i.e., glucose, in biological fluidsl such as serum and urine, have been advantageous in the diagnosis of disease.
U~S. Patent No. 4/273~868~ commonly assigned herewith, discloses a composition, a test device, a method of makinq the test device and a process for determining glucose in a sample. The test composition comprises glucose oxidase, a peroxidatively active substance such ~s peroxidase, a ~0 stabilizing agent and-~ 3,3',5,5'-tetraalkylbenzidine indicator in an amount suf f iciPnt to rapidly produce; upon contact of the test means with a predetermined amount of a glucose-containing sample, a stable colored re~ction product believed to comprise reduced and ~xidized forms of said indicator in stable equilibrium. Preferably9 3,3',5,~'-tetramethylbenzidine is present in a concentration of at least about 2.6 millimoles per thousand International Units of glucose oxidase activity. One of the disclosed ~tabilizing agents is an interpolymer of methylYinyl ethPr 30 and maleic anhydride~ marketed commerically as Gantrez ~,i ~, MS-1235 C ~
* Trade Mark AN-139 by GAF Corporation. The tes-t devices are prepared by a two-dip impregnation process where the 3,3 ', 5,5'--tetraalkylbenzidine is impregnated in the second dip us-ing a solution -thereof prepared in an organic solvent.
U.S. Patent No. 4,361,648 discloses an improvement thereover wherein a carrier is impregnated with an aque-ous solution of tetraalkylbenzidine dihydrochloride and polymeric mordant and dried, then impregnated with a solution of a glucose oxidase and peroxidase, and then impregnated with a solution oE a film-forming agent in a volatile solvent. Such test strip i5 quite satisfactory for the instrumental determination oE glucose levels from 0-4000 milligrams per liteE (mg/liter). Such strip, how-ever, is not suitable for visual determination of rela-tively high glucose levels since the color differences are inadequate.
Barham and Trinder in Analyst, February 1972, Vol.
97, pages 142-145, Trinder in Ann. Clin. Biochem, 6 (1969) 24-27, and Fossati et al in Clin. Chem, 26/2 (1980) 227-231 discuss using 4-aminoantipyrine as a color coupler with 3,5-dichlorohydroxybenzensulfonic acid in determining glucose levels using glucose o2~idase/perox-idase system. This test is a liquid assay and is used primarily for glucose concentration between 0-4000 mg/
:Liter. In this test, whole blood is added to a protein precipitant solution and centrifuged. The clear fluid is added to the color reagent, incubated at 30C for 15 minutes and then the optical density is read at 515 nan~
ometers ~nm) by a spectrophotometer. This liquid system results in expected kinetics and is slow.
It is accordingly an object of the invention to pro-vide an improved test strip which involves an atypical kinetic system, reacts quickly to permit visual quanti-tative glucose determination over the range from about 2000 to 8000 mg/liter, and which strip is simple and in-expensive to prepare.

~. ~.#~

SUM~IARY OF TEIE :[NVENTION

These and other objects and advantages are realized in accordance with the present invention pursuant to which there is provided an analytical element prod~lced by the 5 steps of (a) impregnating a carrier with a firs-t solution, having dissolved therein 4-am:inoantipyrine, or a salt thereof, and 3 hydroxy-2,4,6~triiodo~benzoic acid or

3,5~dichloro-2-hydroxy-ben~ene sulfonic acid, or salts 10 ~hereof, a glucose oxidase and a peroxidase, and dryin~ the carrier; and (b3 applying to the carrier a second solution of a film-forming agent in a volatile solvent, and drying to remove the volatile solvent and leave a f1lm over the dried 15 first impregnant.-In accordance with another aspect of the invention,such an element is used in conjunction with a conventional second element which quantifies glucose contents below about 1800 mg/liter. Thus, if the second element indicates a 20 ylucose level beyond its xange the technician -then uses the fi~st element.
Likewise there i5 provided an analytical element and method for the determination of an analyte in a fluid sample which comprises contacting a sample with the analytical 25 element according to the invention and observing any resultant color change. The colored reaction product is produced within a time period of about 60-90 seconds after contact of the analytical element with the body fluid sample to be tested.

DESCRIPTION OF T~IE PRE~F~RRED EMB~DIMENTS

Although specific terms are used in the following description for the sake of clarity, these terms are intended to refer only to the particular embodiment of the 5 invention selected for exemplary illustration, and are no-t intended to define or limit the scope of the invention.

ANALYTE~RESPONSIVE COMPONENT

The analyte-responsive component comprises those reagents which interact with the analyte and/or resulting 10 products thereof to produce an oxidizing substance, e.g., hydrogen peroxide. In the presence of a peroxidatively active substance, which is one reagent of the analyte-responsive component, the oxidizing substance oxidizes the indicator to produce a detectable species 15 thereof.
rrhe indicator coupling agent comprises 4-aminoanti-pyrine~AAP) and 3,5-dichloro-2-hydroxy-benzene sulfo~ic acid and salts thereof. As an alternative, 3-hydroxy~2,4,6-triiod~benzoic acid (mw 515.8) can be 20 su~stituted for the 3,5-dichloro-2-hydroxy-benzene sulfonic acid.Attempts to use tetraalkylbenzidines as the indicator were unsuccessful because they lack the necassary reaction kinetics properties. 3-Methyl-2-benzothiazolinone hydrazone(MBTH3 with primiquine diphosphate was unsuitable.
25 4-aminoantipyrine with coupling agents 3-hydroxy-1,2,3,4-tetrahydrobenzo(n)quinoline(HTBQ~, methyl catechol, primiquine diphosphate and phenothiazine was also unsuitable.
The combination of 4-aminoantipyrine and 3,5-dichloro 30 2-hydroxy-benzene sulfonic acid, and the sodium or potassium salts thereof, performs successfully and best at particular ratios, as well as enzyme levels, buffer concentrations and polymer mo:rdants to monitor the color formation so that 3~

essentially no color is genera-ted with a glucose concentration less than 1800 mg/liter and then a sliyht peach color starts to form around 2000 mg/liter and continues to produce more color until 8000 mg/liter. As a result, good color resolution exists in every adjacent color block~

POLYMERIC MORDANT
Suitable polymeric mordants which can be used include poly(carboxylic acids) having the formula:
lo t (c~ c ~o ~ / n wherein R is a C1 Cl8 alkyl or amide, and n is an in-teger from 2 to the total number o~ repeating units of the polymer. Examples include polyacrylic acid and polyacrylamide copolymer.
Other polymeric mordants which can be used are copolymeric anhydrides having tne formula:
H R H H
C = C~ C = C
' O = C C ~
/
~1 wherein R i5 C1-Cl8 alkyl, ether, acetate or benzyl and n is an integer from 2 to the total number of repeating units of the polymer. Examples-include methyl vinyl e-ther maleic anhydride copolymer; vinyl acetate-maleic anhydride copolymer; ethylene-maleic anhvdride copo]ymer; octadecyl vinyl ether-maleic anhydride copolymer; and styrene maleic anhydride copolymer.

The polymeric mordant is preferably present in a concerltration o~ from about 0.5 to about 0.75 percen-t of the composition on a weight -to weight basis.

CARRIER

The term carrier refers -to matrices which are in-solu~le in and maintain their structural in~egrity when exposed to physiological or other liquid to be tested.
Suitable matrices which can be used include paper, cel-lulose, wood, synthetic resin fleeces, glass -Eiber, non-woven and woven fabrics, gelatin, various organic poly-mers, such as polypropylene/ and other organic materials well known as film ormers to those skilled in the art.
For convenience, th~ carrier can be suitably attached to an insoluble support or handle member which can be made from polystyrene~

ELEMENT PREPARATION

~s recited hereinabove, the test device is prepared by a process which comprises impregnating the carrier with a first solution having dissolved therein the indica-tor-coupling agent and a glucose oxidase-peroxidase. The glu-cose oxidase-peroxidase is described more fully in U.S.
Patent No. 4,361,648.
Using Whatman 3MM paper, the concentration of the 4-aminoantipyrine or salt thereof in the first solution is from about 5 to 40, preferably about lO to 30, and most preferably about 15 millimoles/liter(mmoles/liter), while the concentration of the 3,5-dichloro-2-hydroxy-benzene sulfonic acid or salt thereoE is from abou-t 0.5 to 500, preferably about 1 to 50, and mos-t pre~erably about 6 mmoles/liter.

* Trade Mark "~

The solution is advan~acJeously buEfered -to a p~l of ,about 5 to 8, preEerab~y about 7, employing a known noninterfering bu~fer such as tris~malonate. Citrate or any other buffer which will provide the same pH range can be 5 used.
Drying is of course eEfected as quickly as possible.
The second step in element preparation comprises impregnating the carrier with a solution of a semi-permeable polymer, such as ekhyl cellulose, in an organic solvent and 10 drying the carrierO The or~anic solvent preferably includes toluene. Particularly pref~rred is an organic solvent which includes toluene and ethanol. Where the solvent conslsts essentially of toluene ~nd ethanol, the toluene is Erom about 80 to 95 percent of the solvent and the ethanol is 15 from about 5 to a~out 20 percent of the solvent, the toluene and ethanol being together 100 percent on a volume/volume basis~
Suitable film-Eorming agents include other hydrophobic cellulose ethers and esters, the film serving to prevent 20 formed eleme,nts (red blood cell9~ from absorbing into the carrier during the period of use.

AN~LYTICAL PROCEDURE

The test device is advantageously used b,y momentarily dipping it in a test sample or by otherwise introducing a 25 test sample onto the carrier matrix, whereby a de-tectable color changè results when glucose is present. The volumetric capacity of khe carrier serves to limit the amount of sample absorbed thereby and to which the kest means incorporated therewith is exposed. Any excess sample 30 can be removed by washing the carrier to thereby limit the amount of sample tested to the volume thereof which has actually entered the carrier matrix. The liqui~ medium to M'~ 35 be assayed can be a naturally occurring or artificially formed liquid suspected to contain ~he ligand, and usually is a biologoical fluid or dilution thereof. siological _ fluids -that can be assayed includ,e serum, plasma, urine, 5 saliva, and amniotic and cerebrospinal fluids. The tes-t device can be used in the same way when samples of plasma, serum or o-ther body fluids are tested.
Semi quantitative results can be obtained using the analytical, elemen-t of the p~esen-t :inVentiOIl by compa-rlng tlle 10 color produced with a panel of standard colors ohtained with known concen-trations o~ analyte employing the same indicator~
The key issue invo~ving the chemistry of the novel elemen~s is -that i~ yields reaction kinetics which are lS atypical compared to tes-t systems with other indicators in solid or liquid-phase; and even the same components in liquid-phase or in solid-phase with a different composition.
Because of these a-typical kinetics, the elen~ent is unresponsive to glucose levels below about 1500 mg/liter, 20 and so is still quite light in color at 2000 mg/liter, the beginning of its intended use range; above this level, typical reaction kinetics occur, and higher glucose levels are readily quantitated. In contrast, any other system which displays more typical reaction kinetics will either 25 (a) be so dark at high'glucose levels that visual quantitation at high levels is difficult, or (b) be so unreactive in order still to be light in appearance at glucose levels of about 2000 mg/liter that again it will not - yield easily quantitated visual differences at various high 30 levels.
EXAMPLE

The example shown is merely illustratlve and not to be construed as a limit~tion of the invention. One skilled in the art will be able to make such variations, substitutions 35 and changes in the ingredients and parameters as may seem desirable. Peroxidase ~9105MR-horseradish)and glucose ~t~3~ L

oxidase (9400MRfrom Aspergillus niger) used in the e~amples were obtained from the Research Produc-ts Division, Mlles Laborator:ies, Inc., Elkhar-t/ IN. Gantrez AN 139 was obtained from GAF Corp., Chemical Products, ~.Y., N.~.
5 The activity of the enzyme preparation is measured by the number of units of activity per milligram of dry weight.
~he Commission on Enæymes of the International Union Oe Biochemistry has defined an International Unit (I.U.) of enzyme activity as 1 micromole (umol) of substrate utili~ed 10 per minute under specified ¢onditions of pll and temperature contro1.
Example I - Analytical Element for Glucose Assay In the experiments reported by this example arl analytical element was preparec~ by the method according to 15 the invention and tes~ed for its ability quantitatively to determine visually the presence of glucose in a liquid sample. Gantrez AN-139, a polycarboxylic anion (chemically it is the interpolymer of m@thyl vinyl ether and maleic anhydride), was added in the first dip. The Gantrez behaves 20 as a dye mordant, so forminy a complex, in the system, thereby protecting final colored reaction productO Ethyl cellulose in toluene was used as the second dip.
El~ment Preparation The solutions used in preparing the glucose specific 25 element contained the following components:
First Dip, for every 100 millilters (ml)

4-AAP 0.36 grams ~g) Gantrez (2%~ 25 ml Tris-~alonate Buffer,pH-~7.4 20 ml ~2 55 ml 3,5-Dichloro-2-hydroxy-benzene sulfonate sodium salt 0.12 g Peroxidase 0.2 g Glucose Oxidase (1000 IU/l) 6.0 ml Second Dip, for every 100 ml Ethocel (ethyl cellulose~ 1.5 g Toluene 95 ml Ethyl Alcohol S m]
MS-l2~5 ~ t,~

Reagent-contain,in~ Whatman 3MM filter paper (Whatman, Inc.~ Clifton, N.J.) is prepared by ~a) irnpreynatiny sheets of the paper to saturation with -thP first ~olu-tion and drying the paper at 60~ Centigrade (C) for 10 minutes; Ib) impregnating the paper of (a) to saturation with the second solution and drying at 40~C for 10 minutes.
The reagent-containing paper was cut to 0.5 cm ~centimeter) x 1.0 cm ~imensions and fixed to one end of a 0.5 cm x 8.25 cm polystyrene ilm by double-faced adhesive 10 tape, providing devices acc~r~ing to the invention. These were stored with a dessicant in brown glass bottles untll used.

Test Solution~

Fresh blood collected into evacuated collection tubes 15 containing ethylene diamine tetraacetic acid (EDTA~ was metabol,ically depleted of glucose by incubation at 37C
overnight ~16 - 20 hours). The hematocrit was adjusted to about 45%. Various glucose levels were prepared by adding various amoun~s of stock glucose (10% w/v) into the blood 20 samples.

Analytical Procedure The performance of the reagent device pxepared and incubated as above-describ~d was analyzed by the following procedure:
(a) A large drop of capillary or venous blood sufficient to cover each reagent area i~
applied to the test device.
(b~ Sixty seconds are allowed to elapse~
(c) Each reagent area is then ~ashed with water ~ufficien~ly to remove the blood sample.

M~

(d) Each reagent area is then blotted with a link-free paper towel.
(e) The low range pad or reagent(area) is then compared with color blocks in the range of 200 to 1,800 mg/liter. The color blocks for this range have a greenish, tint and if the color procluced falls betwe!en two colcr blocks the value is interpolated. If the color matche~ or exceeds the color of the 1,800 mg/liter color block, another 30 second~ is allowed to elapse before comparing the resulting color of the high range pad or reagent area with color blocks fcr the high range (2,000 to 8,000 mg/liter), ~5 again the interpolating if the color produced falls between two color blocks The high range color blocks have a peach or orange tint in contrast to the green tint of the low range color blocks.

20 Example II
Another suitable analytical element for the high range glucose levels was prepared by substitutiny in Example I
0.06g o 3-hydroxy-2,4,6-triiodobenzoic acid ~or the 3,5-dichloro-2-hydroxy-benzen sulfonate, sodium salt and substituting 0.18g of 4-AAP HCl for the 4-AAP. Essentially equivalent results were obtained.

It will be understood that the specification and examples are illustrative buk not limitative of the present invention an~ ~hat other embodiments within the spirit and scope oE the invention will suggesk themselves to khose

5 skilled in the art.

Claims

The embodiments of the invention in which an exclu-sive property or privilege is claimed are defined as fol-lows:

1. A method for preparing an analytical element for determining glucose in a liquid sample which method com-prises the steps of a) impregnating a carrier with a first solution hav-ing dissolved therein 4-aminoantipyrine or a salt thereof and 3-hydroxy-2,4,6-triiodobenzoic acid or 3,5-dichloro-2 hydroxy-benzene sulfonic acid, or a salt thereof, a glucose oxidase and a per-oxidase, and drying the carrier; and b) applying to the carrier a second solution of film-forming agent in a volatile solvent, and drying to remove the volatile solvent and leave a film over the dried first impregnant.

2. A method according to claim 1, wherein the con-centration of the 4-aminoantipyrine or salt thereof in the first solution is from about 5 to 40 mmoles/liter.

3. A method according to claim 1, wherein the con-centration of the 3-hydroxy-2,4,6-triiodobenzoic acid or 3,5-dichloro-2-hydroxy-benzene sulfonic acid, or salt thereof, in the first solution is from about 0.5 to 500 mmoles/liter .

4. A method according to claim 1, wherein the film-forming agent in the second solution is a hydrophobic cel-lulose ether or ester.

5. A method according to claim 1, wherein the first solution contains a buffer for maintaining a pH from about 5 to 8 and a mordant.

6. A method according to claim 5, wherein the con-centration of the 4-aminoantipyrine or salt thereof in the first solution is from about 10 to 30 mmoles/liter and the concentration of the 3,5-dichloro-2-hydroxy-benzene sulfonic acid or salt thereof in the first solu-tion is from about 1 to 50 mmoles/liter.

7. A method according to claim 5, wherein the con-centration of the 4-aminoantipyrine or salt thereof in the first solution is about 15 mmoles/liter, the concen-tration of the 3,5-dichloro-2-hydroxy-benzene sulfonic acid or salt thereof in the first solution is about 6 mmoles/liter, and the film-forming agent in the second solution is a hydrophobic cellulose ether or ester.

8. A method according to claim 5, wherein the 4-aminoantipyrine is present in the first solution as the hydrochloride, the sulfonic acid is present in the first solution as the sodium salt, the solvent in the first sol-ution is water, the pH of the first solution is about 7 and it contains a polymeric polycarboxylic acid or anhy-dride thereof as the mordant, and the film-forming agent in the second solution is ethyl cellulose.

9. An analytical element for determining whole blood glucose produced by the process of claim 1.

10. A method for determining the glucose content of whole blood within the range of about 2000 to 8000 mg/liter comprising contacting the whole blood with an analytical element produced by the process of claim 1, and matching the resulting color of the element with predetermined standards.

11. A system for determining the glucose content of whole blood over a range from about 0 to 8000 mg/liter comprising, in combination, an element for identifying a glucose content of up to about 2000 mg/liter, and the an-alytical element produced by the process of claim 1.